Project Summary New imaging tools are needed for brain-wide visualization of the neural activity underlying defined behaviors. Live imaging techniques such as fMRI and MEG measure activity across the brain, but these methods cannot resolve single cell activity. Moreover, in vivo electrophysiology and calcium imaging can reach cellular resolution, but only simultaneously survey a few brain regions at best. Capturing a brain-wide snapshot of recent neural activity at cellular resolution would provide an incredible tool for hypothesis generation and assay development for CNS drug discovery. To date, immunohistochemical imaging of the activity-dependent immediate-early gene (IEG) cFos is the only established platform that visualizes neural activity linked to a defined behavior at cellular resolution. To efficiently collect brain-wide snapshots of neuronal activity, two obstacles need to be overcome. First, since standard histological methods and modern imaging approaches (e.g., serial two-photon) require tissue slicing, these approaches to IEG imaging are time consuming and introduce anatomical distortions that prevent quantitative comparisons among brain regions. Second, since traditional IEGs like cFos are also regulated by paracrine signaling factors, the fidelity of IEG expression as a read-out of neuronal activity is compromised. To increase the throughput of whole-brain histological imaging, we will take advantage of recent developments in tissue clearing, fluorescence light sheet imaging and computational alignment and cell detection. For tissue clearing, we optimized the iDISCO tissue clearing method for whole brain immunostaining. For imaging, we have customized a commercial light sheet micrscope for large format imaging of iDISCO cleared tissue. With our adapted technology, we are imaging a whole mouse brain in less than three hours at higher spatial resolution than in previous reports. Using these methods, we have generated whole brain 3D images of genetically-encoded fluorophores. We will couple this technology to the use of a novel, high quality monoclonal antibody against Npas4, a unique IEG that is neuron-specific and exquisitely tuned to neuronal activity. Our automated quantification pipeline aligns samples to a common reference atlas and generates brain-wide cell counts of neuronal activity across >1,000 identified brain areas. This new imaging platform will overcome the time- and labor-intensive limitations of standard histology, and the use of Npas4 as a marker will provide the highest fidelity, brain-wide read-outs of neuronal activity to date.